- Volume 165, Issue 6, 2019

Assessing bacterial contamination in environmental samples is critical in determining threats to public health. The classical methods are time-consuming and only recognize species that grow easily on culture media. Viable but non-culturable (VBNC) bacteria are a possible threat that may resuscitate and cause infections. Recent dye-based screening techniques employ nucleic acid dyes such as ethidium monoazide (EMA) and propidium monoazide (PMA), along with many fluorescent dyes, which are an effective alternative for viability assessment. The measurement of cellular metabolism, heat flow and ATP production has also been widely applied in detection approaches. In addition, RNA-based detection methods, including nucleic acid sequence-based amplification (NASBA), have been applied for bacterial pathogen determination. Stable isotope probing using 13C, 15 N and 18O, which are mobilized by microbes, can also be used for effective viability assessment. Future detection tools, such as microarrays, BioNEMS and BioMEMS, which are currently being validated, might offer better microbial viability detection.

In natural environments contaminated by recalcitrant organic pollutants, efficient biodegradation of such pollutants has been suggested to occur through the cooperation of different bacterial species. A phenanthrene-degrading bacterial consortium, MixEPa4, from polluted soil was previously shown to include a phenanthrene-degrading strain, Mycobacterium sp. EPa45, and a non-polycyclic aromatic hydrocarbon (PAH)-degrading strain, Burkholderia sp. Bcrs1W. In this study, we show that addition of phenanthrene to rich liquid medium resulted in the transient growth arrest of EPa45 during its degradation of phenanthrene. RNA-sequencing analysis of the growth-arrested cells showed the phenanthrene-dependent induction of genes that were predicted to be involved in the catabolism of this compound, and many other cell systems, such as a ferric iron-uptake, were up-regulated, implying iron deficiency of the cells. This negative effect of phenanthrene became much more apparent when using phenanthrene-containing minimal agar medium; colony formation of EPa45 on such agar was significantly inhibited in the presence of phenanthrene and its intermediate degradation products. However, growth inhibition was suppressed by the co-residence of viable Bcrs1W cells. Various Gram-negative bacterial strains, including the three other strains from MixEPa4, also exhibited varying degrees of suppression of the growth inhibition effect on EPa45, strongly suggesting that this effect is not strain-specific. Growth inhibition of EPa45 was also observed by other PAHs, biphenyl and naphthalene, and these two compounds and phenanthrene also inhibited the growth of another mycobacterial strain, M. vanbaalenii PYR-1, that can use them as carbon sources. These phenomena of growth inhibition were also suppressed by Bcrs1W. Our findings suggest that, in natural environments, various non-PAH-degrading bacterial strains play potentially important roles in the facilitation of PAH degradation by the co-residing mycobacteria.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P_1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H₂O₂ concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

Phenotypically heterogeneous but genetically identical mycobacterial subpopulations exist in in vitro cultures, in vitro-infected macrophages, infected animal models and tuberculosis patients. In this regard, we recently reported the presence of two subpopulations of cells, which are phenotypically different in length and buoyant density, in mycobacterial cultures. These are the low-buoyant-density short-sized cells (SCs), which constitute ~10–20 % of the population, and the high-buoyant-density normal/long-sized cells (NCs), which form ~80–90 % of the population. The SCs were found to be significantly more susceptible to rifampicin (RIF), isoniazid (INH), H₂O₂ and acidified nitrite than the NCs. Here we report that the RIF-/INH-/H₂O₂-exposed SCs showed significantly higher levels of oxidative stress and therefore higher susceptibility than the equivalent number of exposed NCs. Significantly higher levels of hydroxyl radical and superoxide were found in the antibiotic-exposed SCs than in the equivalently exposed NCs. Different proportions of the subpopulation of SCs were found to have different levels of reactive oxygen species (ROS). The hydroxyl radical quencher, thiourea, and the superoxide dismutase mimic, TEMPOL, significantly reduced hydroxyl radical and superoxide levels, respectively, in the antibiotic-exposed SCs and NCs and thereby decreased their differential susceptibility to antibiotics. Thus, the present study shows that the heterogeneity of the reactive oxygen species (ROS) levels in these mycobacterial subpopulations confers differential susceptibility to antibiotics. We have discussed the possible mechanisms that can generate differential ROS levels in the antibiotic-exposed SCs and NCs. The present study advances our current understanding of the molecular mechanisms underlying antibiotic tolerance in mycobacteria.

Regulators encoded by the beta-glucoside (bgl) operon of Escherichia coli are known to influence the expression of downstream target genes that confer a fitness advantage in stationary phase. We have examined the role of bglG in the regulation of ridA that encodes an enamine/imine deaminase essential for the elimination of reactive intermediates generated during the catabolism of amino acids such as serine. We report here that ridA is positively regulated by leucine responsive regulatory protein (Lrp) and leucine antagonizes the activation by Lrp. We also show that Lrp itself is under the indirect regulation of BglG, which brings about the overexpression of ridA in Bgl+ strains during stationary phase. Loss of ridA function in a Bgl+ background results in a significant growth retardation in serine-containing media compared to that in a Bgl− background. We propose that overexpression of ridA in Bgl+ background during stationary phase is physiologically relevant to eliminate toxic metabolites generated by the catabolism of serine-containing peptides as a result of elevated levels of their uptake.